CN113462723B

CN113462723B - Retroviral vectors expressing CAR and shRNA and uses thereof

Info

Publication number: CN113462723B
Application number: CN202110754493.8A
Authority: CN
Inventors: 王建勋; 张静; 朱晶晶; 冯娅茹; 李晓瑞; 尚凤琴
Original assignee: Beijing University of Chinese Medicine
Current assignee: Beijing University of Chinese Medicine
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2023-07-11
Anticipated expiration: 2041-07-05
Also published as: CN113462723A

Abstract

The invention provides a retrovirus vector expressing a CAR and shRNA, wherein the shRNA targeting LSD1 in the retrovirus vector is co-expressed with the CAR. According to the invention, LSD1shRNA and anti-CD19CAR are co-expressed in the CAR-T cells, so that synchronous regulation of the LSD1shRNA on the functions of the CAR-T cells is realized, and a combined treatment method is constructed. In-vivo and in-vitro function verification is carried out on the LSD1shRNA anti-CD19CAR-T cell, and the LSD1shRNA anti-CD19CAR-T cell has better cell activity, proliferation capacity and anti-tumor capacity in-vivo and in-vitro.

Description

Retroviral vectors expressing CAR and shRNA and uses thereof

Technical Field

The invention relates to the technical field of tumor treatment, in particular to an expressed Chimeric Antigen Receptor (CAR) and shRNA retrovirus vector and application thereof.

Background

Short hairpin RNAs (shrnas) belong to the category of RNA interference (RNAi), and after transduction or transfection of cells, precursor shrnas are synthesized in the nucleus, whose sense and antisense strands form stem regions by base pairing, with intermediate unpaired nucleotides forming loops, the entire structure being a hairpin structure. After being processed by ribonuclease IIIDrosha and DGCR8, the siRNA is transported into cytoplasm by export-5 protein, and then is cut by ribonuclease IIIDicer and TRBP/PACT to remove the sequence of the ring structure, thus forming the siRNA. After the siRNA recognizes and integrates with RISC, unwinding occurs and one RNA strand in the double strand is removed, and then the target mRNA is recognized and occupied by the base complementary sequence, resulting in its degradation. The shRNA can be stably expressed in a transduction mode, specifically targets and inhibits the expression of target mRNA, and has wide application in treatment, diagnosis and scientific research. Currently, shRNA which specifically targets an immune checkpoint receptor is coexpressed with CAR-T cells in a slow virus vector transduction mode to inhibit the expression of the immune checkpoint receptor and inhibit the tumor microenvironment, so that immune response is regulated.

LSD1 is a flavin-dependent monoamine oxidase, also the first histone lysine-specific demethylase discovered to participate in the demethylation of either monomethylated or dimethylated H3K4 or H3K9 in the presence of xanthine-adenine dinucleotide, regulating the activation or inhibition of gene transcription in different environments from epigenetic modifications, participating in different physiological processes including hematopoiesis, lipogenesis, developmental processes, etc., and also playing an important role in the development of tumors, LSD1 inhibitors are potential antitumor immunosuppressants. Inhibiting LSD1 can stimulate the immunogenicity of tumors, stimulate interferon-dependent anti-tumor immunity, and promote T cell infiltration. There are many LSD1 inhibitors currently undergoing clinical evaluation for cancer treatment.

Disclosure of Invention

In order to solve the technical problems, the invention provides a novel combined treatment method for coexpression of shRNA and CAR in CAR-T cells.

In one embodiment, a retroviral vector is provided that expresses a CAR and an shRNA, wherein the shRNA targeting LSD1 is co-expressed with the CAR in the viral vector.

In one embodiment, the U6 promoter and EF 1a promoter in the viral vector are integrated into the CAR expression vector, respectively, instead of the long terminal repeat driving expression, the EF 1a promoter driving expression of LSD1shRNA by the U6 promoter drives expression of an anti-CAR, preferably an anti-CD19CAR and/or CD38 CAR.

In one embodiment, the retroviral vector comprises, in series, a U6 promoter, an LSD1shRNA, an EF1 alpha promoter, an upstream signal peptide, and a myc tag for detection; a CD19CAR antigen binding region; CD8 hinge-transmembrane domain; CD28 or 4-1BB synergistic activation domain and cd3ζ intracellular signaling domain.

In one embodiment, the shRNA targeting LSD1 has clone ID TRCN0000046068, clone designation nm_015013.1-1812s1c1, sequence SEQ ID No. 2 gcctagatataaactgaata.

In one embodiment, the clone ID of the shRNA targeting LSD1 is TRCN0000046069, the clone name is NM_015013.1-2168s1c1, and the sequence is SEQ ID NO: 3:GCTCCAATACTGTGGCACTA.

In one embodiment, a targeted chimeric antigen receptor T cell is provided that includes a targeted chimeric antigen receptor expressed by the retroviral vector described above.

In one embodiment, a medicament for treating a tumor is provided comprising the chimeric antigen receptor T cell described above.

In one embodiment, the tumor is multiple myeloma.

In one embodiment, there is provided the use of a retroviral vector as described above, wherein a gene fragment encoding the chimeric antigen receptor is inserted into the vector, packaged into viral vector particles, and infected with human T cells to produce chimeric antigen receptor T cells for use in the treatment of surface CD 19-positive and/or CD 38-positive tumors.

In the invention, a genetic engineering means is adopted to construct MFG (retrovirus vector plasmid) -LSD1 shRNA-anti-CD19 CAR retrovirus vector plasmid, a U6 promoter, LSD1shRNA and EF1 alpha promoter are integrated into a retrovirus vector for expressing the CAR, the U6 promoter drives the expression of the LSD1shRNA, and the EF1 alpha promoter drives the expression of the anti-CD19 CAR. Packaging the retroviral vectors to obtain two LSD1shRNA anti-CD19CAR retroviral vectors with higher titer, transducing human primary T cells, and successfully constructing the LSD1shRNA anti-CD19CAR-T cells, wherein the transduction efficiency of the detection by flow cytometry is more than 50%. qPCR detects that the expression level of LSD1 in LSD1shRNA anti-CD19CAR-T cells is obviously reduced, which indicates that LSD1shRNA can be expressed simultaneously with the CAR genes, and the simultaneous expression of LSD1shRNA does not affect the expression of anti-CD19 CAR.

LSD1shRNA enhances anti-CD19CAR-T cell in vitro anti-tumor function. Through flow cytometry apoptosis detection, luciferase detection and RTCA detection, LSD1shRNA can enhance the function of anti-CD19CAR-T cells in vitro to kill tumor cells; pressure test experiments prove that LSD1shRNA can promote the long-term anti-tumor function of anti-CD19CAR-T cells under repeated antigen stimulation; proved by ELISA detection cytokine experiments, the LSD1shRNA can promote the release level of IFN-gamma, TNF-alpha and IL-2 when the anti-CD19CAR-T cells kill tumor cells, and promote the anti-tumor function of the anti-CD19CAR-T cells; cell proliferation fold and CFSE proliferation detection are calculated through cell counting, and the LSD1shRNA can enhance the in vitro proliferation capacity of anti-CD19CAR-T cells.

LSD1shRNA enhances anti-tumor function in anti-CD19CAR-T cells. NOD-Prkdc using immunodeficiency ^scid Il2rg ^null After constructing a Raji-Luc cell tumor animal model by an NPG mouse, performing in vivo imaging, weight monitoring, peripheral blood T cell flow cytometry detection and other methods on a small animal after LSD1shRNA anti-CD19CAR-T cell treatment, proving that LSD1shRNA anti-CD19CAR-T cells show good anti-tumor function in vivo, completely eliminating tumors, obviously prolonging the survival time of the tumor model mouse, and although LSD1shRNA anti-CD19CAR-T cell treatment groups and RNAU6 anti-CAR-T cell treatment groups have no obvious difference in tumor bioluminescence signals and survival rate of the small animal in vivo imaging, ELISA detection on mouse serum IFN-gamma after 7 days after the second injection of the CAR-T cells finds that the serum IFN-gamma level of the LSD1shRNA co-expressed anti-CD19CAR-T cell treatment groups is obviously increased, and LSD1shRNA enhances the anti-tumor function in vivo of the CAR-T cells. At the end of the study on day 52, flow cytometry detected significantly higher numbers of T cells in peripheral blood of mice of the LSD1 shRNA-2 anti-CD19CAR-T cell treatment group than the control group, indicating that LSD1shRNA promoted the proliferation capacity of CAR-T cells in vivo. Therefore, LSD1shRNA overexpression promotes anti-tumor activity and proliferation capacity of anti-CD19CAR-T cells in vivo.

In the invention, LSD1shRNA and anti-CD19CAR are co-expressed in CAR-T cells, so that synchronous regulation of the LSD1shRNA on the function of the CAR-T cells is realized, and a combined treatment method is constructed. In-vivo and in-vitro function verification is carried out on the LSD1shRNA anti-CD19CAR-T cell, and the LSD1shRNA anti-CD19CAR-T cell has better cell activity, proliferation capacity and anti-tumor capacity in-vivo and in-vitro.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of different CAR expression vectors, wherein FIG. 1A is a schematic diagram of an unmodified anti-CD19CAR expression vector, FIG. 1B is a schematic diagram of a modified control U6 anti-CD19CAR expression vector, and FIG. 1C is a schematic diagram of a U6-LSD1 shRNA-anti-CD19 CAR expression vector;

FIG. 2 is a graph of the results of a test for efficiency of transfection of a retroviral vector expression plasmid into Phoenix-ECO cells, wherein FIG. 2A is pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR and FIG. 2B is pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19 CAR;

FIG. 3 is a graph of the results of a test for the efficiency of transduction of PG13 cells by an avirulent retroviral vector, wherein FIG. 3A is an LSD1 shRNA-1 anti-CD19 CAR; FIG. 3B is an LSD1 shRNA-2 anti-CD19 CAR;

fig. 4 is a graph of results of detection of LSD1 expression levels in LSD1shRNA anti-CD19CAR-T cells (n=3), wherein fig. 4A is detection of LSD1 expression levels in LSD1 shRNA-1 anti-CD19CAR-T cells; fig. 4B is LSD1 shRNA-2 anti-CD19CAR-T cell LSD1 expression level detection, P <0.05, < P <0.01 compared to RNAU6 anti-CD19CAR-T cells;

fig. 5 is a graph of results of in vitro killing Raji cell efficiency assays (n=3) of LSD1 shRNA-1 anti-CD19CAR-T cells, P <0.05 compared to RNAU6 anti-CD19CAR-T cells;

fig. 6 is a graph of the results of pressure test detection of CAR-T cell killing following in vitro repeated antigen stimulation, wherein 6A: cell killing efficiency detection after the first co-culture period; 6B: cell killing efficiency detection after the second co-culture period; 6C: cell killing efficiency detection after the third co-culture period; p <0.05, < P <0.01 compared to RNAU6 anti-CD19CAR-T cells;

FIG. 7 is a graph of LSD1shRNA anti-CD19CAR-T cell killing Raji-Luc cell efficiency detection results, compared with RNAU6 anti-CD19CAR-T cells, P <0.05;

fig. 8 is a graph of LSD1shRNA anti-CD19CAR-T cell killing SW620 cell efficiency assay (n=3), wherein fig. 8A is SW620 cell index variation; figure 8B is the SW620 cell index at the experimental termination time intercept point,

(P <0.001 compared to RNAU6 anti-CD19CAR-T cells);

FIG. 9 is a graph showing results of detection of levels of release of LSD1shRNA anti-CD19CAR-T cell cytokine (n=3), wherein 9A is the level of IFN- γ release in the supernatant of co-cultured cells; 9B is TNF- α release level in the supernatant of co-cultured cells; 9C is the level of IL-2 release in the supernatant of co-cultured cells (P <0.05, < P <0.01 compared to RNAU6 anti-CD19CAR-T cells);

FIG. 10 is a graph of LSD1shRNA anti-CD19CAR-T cell proliferation recordings, where 10A is a cell proliferation curve and 10B is a cell viability curve;

FIG. 11 is a graph of the detection results of CD4+ T cells and CD8+ T cells in CAR-T cells;

fig. 12 is a graph of results of tumor bioluminescence signal intensity monitoring (n=6) of in vivo imaging of small animals, 12A being tumor area change, 12B being overall signal intensity of tumor bioluminescence;

fig. 13 is NPG mouse survival curve (n=6);

fig. 14 is a NPG mouse body weight change curve (n=6);

fig. 15 results of serum IFN- γ release level detection (n=6) graph, P <0.05, P <0.01 compared to RNAU6 anti-CD19CAR-T cell treatment group.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.

EXAMPLE A CAR Structure and plasmid map of the invention

The invention applies LSD1shRNA to CAR-T cell engineering to construct a novel combined treatment method, and integrates the LSD1shRNA into a retrovirus vector expressing the CAR and simultaneously regulates gene expression. To ensure efficient inhibition of target mRNA, two shRNA targeting different sequence targets of LSD1 mRNA were selected. To enable better expression of LSD1shRNA and CAR, the U6 promoter and EF 1A promoter were integrated into the CAR expression vector, respectively, instead of the Long Terminal Repeat (LTR) to drive expression, the U6 promoter was used to drive expression of LSD1shRNA, and the EF 1A promoter was used to drive expression of anti-CD19CAR to extend expression of CAR in T cells, see fig. 1, where fig. 1A is a schematic diagram of an unmodified anti-CD19CAR expression vector, fig. 1B is a schematic diagram of an engineered control U6 anti-CD19CAR expression vector, and fig. 1C is a schematic diagram of a U6-LSD1 shRNA-anti-CD19 CAR expression vector. MMLV (truncated) in FIG. 1 is MMLV-encoded retroviral helper DNA. The anti-tumor function of LSD1shRNA anti-CD19CAR-T cells is evaluated through in vitro and in vivo function detection. Provides a new research thought for exploring the activity improvement and the function optimization of the CAR-T cells, provides a new method for treating multiple myeloma, and lays an experimental foundation for the treatment of other blood system tumors and solid tumor CAR-T cells. ScFv amino acid sequence of anti-CD19 CAR:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:1)

EXAMPLE two construction of pMFG-U6-LSD1 shRNA-EF1 alpha-anti-CD 19CAR plasmid

LSD1shRNA base sequence search

According to the database and literature search, a human LSD1shRNA target sequence is obtained, and two LSD1shRNA target sequences are selected for constructing pMFG-U6-LSD1 shRNA-EF1 alpha-anti-CD 19CAR plasmids, wherein pMFG represents a retroviral vector plasmid, and the table is shown in Table 1. Referring to the website: https? transcame=nm_ 015013.4.

TABLE 1 LSD1shRNA target sequences and primers

Preparation of LSD1 shRNA-1 fragment

Designing a primer, introducing a restriction enzyme site AgeI into the 5 'end of the LSD1 shRNA-1 primer, introducing a restriction enzyme site EcoRI into the 3' end, and synthesizing a corresponding primer sequence, wherein the primer synthesis work is completed by a division of biological engineering (Shanghai) Co. Gradually annealing the upstream and downstream primers of the LSD1 shRNA-1 to obtain the LSD1 shRNA-1 fragment, wherein the tail end of the LSD1 shRNA-1 DNA is a sticky tail end. Reaction conditions: 95℃for 5min,92.5℃for 3min,90℃for 3min,87.5℃for 3min … …, decreasing successively by 2.5℃until the temperature drops to 10 ℃.

Preparation of 3U6-LSD1 shRNA-2 fragment

The U6-LSD1 shRNA-2 sequence is synthesized, a restriction enzyme site Mlu I is introduced into the 5 'end of the DNA sequence, a restriction enzyme site Pac I is introduced into the 3' end of the DNA sequence, and the whole DNA sequence is cloned into a PUC57 vector.

Verification of pMFG-U6-LSD1 shRNA-EF1 alpha-anti-CD 19CAR plasmid expression

Recovering HEK-293T cells and seeding in T75 cell culture flasks, passaging HEK-293T cells and seeding in 6 well cell culture plates at 1X 10 per well when the confluence of cells in T75 culture flasks reached about 80% ⁶ A total of 3 wells were seeded with each cell. The cells are marked as pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR control hole, pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR experimental hole, pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR experimental hole, and put into CO at 37 DEG C ₂ An incubator. When the cell confluency reached about 80%, HEK-293T cells were transfected with the expression plasmid using the transfection reagent FuGene HD and placed at 37℃CO ₂ Incubate in incubator for 24h.

After 24h the culture medium supernatant in each group of cell culture plates was carefully removed, discarded and each well was supplemented with 3mL of fresh DMEM complete medium. After 24h the culture medium supernatant from each group of cell culture plates was carefully removed, discarded, after washing the cells with 1 XPBS, the cells were pancreatin digested and collected in 1.5mL EP tubes for cell counting. Each group is 1X 10 ⁶ Individual cells were used for flow cytometry to determine transfection efficiency, taking 2X 10 ⁶ Individual cells were used for total cellular RNA extraction and RT-qPCR to detect LSD1 expression levels.

LSD1 expression level detection

The total RNA of transfected or transduced cells is extracted, the expression level of LSD1 is detected by a qPCR dye method, the GAPDH gene is used as an internal reference gene, the primer sequence is shown in table 2.12, jw389 and jw390 are used as target fragment amplification primers of the LSD1 gene, and the target fragment amplification primers are shown in table 2. Data processing fold changes in LSD1 expression levels were calculated using the 2- Δct method.

TABLE 2 primer sequences for qPCR detection of LSD1 Gene expression level

6. Results

Construction of a plasmid 6.1pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR

The pMD18S-T-U6-RNA plasmid is digested with restriction enzymes Age I and EcoR I, and the RNA sequence in the plasmid is replaced by the LSD1 shRNA-1 sequence to obtain the pMD18S-T-U6-LSD1 shRNA-1 plasmid. Then, the restriction enzyme Pac I and Mlu I-HF are used for double enzyme digestion of pMD18S-T-U6-LSD1 shRNA-1 plasmid to obtain U6-LSD1 shRNA-1 fragment. And replacing the U6-LSD1 shRNA-1 fragment with U6-RNA in the pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR plasmid to obtain the pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR plasmid.

6.1.1 construction of pMD18S-T-U6-LSD1 shRNA-1 plasmid

The restriction enzyme Age I and EcoR I double-cleave the pMD18S-T-U6-RNA plasmid to obtain the pMD18S-T-U6 (Age I/EcoR I) vector. The digested product was subjected to agarose gel electrophoresis and the DNA was gel purified and recovered, and the size of the objective vector was about 3kb.

The LSD1 shRNA-1 fragment was ligated into pMD18S-T-U6 (Age I/EcoR I) vector to obtain pMD18S-T-U6-LSD1 shRNA-1. The procedures of ligation, transformation and plasmid miniprep. are the same. And selecting plasmids with correct enzyme digestion identification, and sequencing, wherein the sequencing primer is a universal primer M13-For. Sequencing results show that the U6-LSD1 shRNA-1 base sequence is completely correct.

6.1.2 construction of pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR plasmid

The restriction enzymes Pac I and Mlu I-HF are used for respectively double-enzyme cutting pMD18S-T-U6-LSD1 shRNA-1 plasmid and pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR plasmid to obtain U6-LSD1 shRNA-1 fragment and pMFG-EF1 alpha-anti-CD 19CAR vector. The digested product is subjected to agarose gel electrophoresis, DNA is purified and recovered, the U6-LSD1 shRNA-1 target fragment size is 337bp, and the pMFG-EF1 alpha-anti-CD 19CAR vector size is about 9.2kb.

And (3) connecting the U6-LSD1 shRNA-1 target fragment into a pMFG-EF1 alpha-anti-CD 19CAR vector to obtain the pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19 CAR. The procedures of ligation, transformation and plasmid miniprep. are the same. And selecting plasmids with correct enzyme digestion identification for sequencing, and the result shows that the plasmids are successfully constructed.

Construction of pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR plasmid

The PUC57-LSD1 shRNA-2 plasmid was digested with the restriction enzymes Pac I and Mlu I-HF to obtain the U6-LSD1 shRNA-2 fragment. And replacing the U6-LSD1 shRNA-2 fragment with U6-RNA in the pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR plasmid to obtain the pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR plasmid.

7.1U6-LSD1 shRNA-2 fragment and pMFG-EF1 alpha-anti-CD 19CAR vector

And respectively carrying out enzyme digestion on the PUC57-LSD1 shRNA-2 plasmid and the pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR plasmid by using restriction enzymes Pac I and Mlu I-HF to obtain a U6-LSD1 shRNA-2 fragment and a pMFG-EF1 alpha-anti-CD 19CAR vector. The enzyme-digested product is subjected to agarose gel electrophoresis, DNA is purified and recovered, the U6-LSD1 shRNA-2 target fragment size is 324bp, and the pMFG-EF1 alpha-anti-CD 19CAR vector size is about 9.2kb.

7.2 construction and identification of pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR plasmid

And connecting the U6-LSD1 shRNA-2 fragment into a pMFG-EF1 alpha-anti-CD 19CAR vector to obtain the pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19 CAR. The procedures of ligation, transformation and plasmid miniprep. are the same. And selecting plasmids with correct enzyme digestion identification for sequencing, and the result shows that the plasmids are successfully constructed.

7.3pMFG-U6-LSD1 shRNA-anti-CD19 CAR plasmid expression detection

HEK-293T cells are transfected by pMFG-U6-LSD1 shRNA-1-anti-CD19 CAR and pMFG-U6-LSD1 shRNA-2-anti-CD19 CAR plasmids, and the transfection efficiency is detected by flow cytometry after 48 hours. The results show that: has higher transfection efficiency. RT-qPCR detects LSD1 expression level changes. The results show that: the expression level of LSD1 in the HEK-293T cell group transfected with the MFG-U6-LSD1 shRNA-1-anti-CD19 CAR (0.49+/-0.044:1) and the expression level of LSD1 in the HEK-293T cell group transfected with the MFG-U6-LSD1 shRNA-2-anti-CD19 CAR (0.79+/-0.003:1) are obviously lower than those in the control group, so that the LSD1shRNA can be expressed normally and can function. Therefore, the next retroviral vector packaging experiment can be carried out, the LSD1shRNA anti-CD19CAR-T cell can be constructed, and in vivo and in vitro function verification can be carried out.

EXAMPLE three LSD1shRNA anti-CD19CAR-T cell construction

1.LSD1 shRNA anti-CD19CAR-T cell construction

Constructing LSD1shRNA anti-CD19CAR-T cells by preparing retrovirus vectors and transducing T cells, using RNAU6 anti-CD19CAR-T cells as a control group, and detecting the transduction efficiency of the CAR-T cells by flow cytometry.

2.LSD1 shRNA anti-CD19CAR avirulence retrovirus vector preparation

And respectively transfecting Phoenix-ECO cells with pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR and pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR expression plasmids to prepare the avidity retrovirus vector. Flow cytometry detection of the expression level of the Myc tag of Phoenix-ECO cells 48h after transfection showed that: the pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR and pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR plasmids were able to be expressed at higher levels in Phoenix-ECO cells, see FIG. 2.

3.LSD1 shRNA anti-CD19CAR amphotropic retroviral vector preparation

The amphotropic retroviral vector respectively transduces PG13 cells, and a PG13 cell line for stably producing the LSD1 shRNA-1 anti-CD19CAR amphotropic retroviral vector and a PG13 cell line for stably producing the LSD1 shRNA-2 anti-CD19CAR amphotropic retroviral vector are established. Flow cytometry detects the transduction efficiency of the eosinophil into PG13 cells by the eosinophil retroviral vector, and the results show that: the transduction efficiency of PG13 cells reaches more than 70%, and referring to FIG. 3, the successful construction of a PG13 cell line for stably producing LSD1 shRNA-1 anti-CD19CAR amphotropic retroviral vector and a PG13 cell line for stably producing LSD1 shRNA-2 anti-CD19CAR amphotropic retroviral vector is demonstrated.

4. Retroviral vector titre detection

The amphotropic retroviral vector is harvested, the titer of the viral vector is detected by RT-qPCR, the retroviral vector is successfully prepared, and the next experiment can be carried out.

5.LSD1 shRNA anti-CD19CAR-T cell construction

PBMCs were isolated from peripheral blood donated from healthy volunteers, T cells cultured and activated in vitro. T cells are transduced by using the LSD1 shRNA-1 anti-CD19CAR amphotropic retroviral vector and the LSD1 shRNA-2 anti-CD19CAR amphotropic retroviral vector respectively, so that the LSD1 shRNA-1 anti-CD19CAR-T cells and the LSD1 shRNA-2 anti-CD19CAR-T cells are constructed, and the transduction efficiency is detected by flow cytometry. The results show that: the transduction efficiency reaches more than 50%, which indicates that simultaneous expression of LSD1shRNA does not affect the expression of anti-CD19 CAR.

6.LSD1 shRNA anti-CD19CAR-T cell integrated copy number detection

qPCR detects integrated copy number of LSD1 shRNA-1 anti-CD19CAR-T cell and LSD1 shRNA-2 anti-CD19CAR-T cell virus vector genes. The results show that the integrated copy number of all CAR-T cell retrovirus vectors is less than 3, the anti-CD19CAR copy number is 2.26+/-0.12, the RNAU6 anti-CD19CAR copy number is 1.81+/-0.03, the LSD1 shRNA-1 anti-CD19CAR copy number is 2.27+/-0.41, and the LSD1 shRNA-2 anti-CD19CAR copy number is 2.31+/-0.25.

Detection of LSD1 expression level

Transduction of LSD1shRNA anti-CD19CAR retroviral vector into human primary T cells LSD1shRNA anti-CD19CAR-T cells were constructed, FITC-labeled CD19 recombinant antigen molecules were used, and flow cytometry detected expression of anti-CD19CAR, results showed: anti-CD19CAR is expressed with high efficiency. The expression level of LSD1 was then examined by RT-qPCR, which showed that: the level of LSD1 expression in LSD1 shRNA-1 anti-CD19CAR-T cells and LSD1 shRNA-2 anti-CD19CAR-T cells was significantly reduced, see FIG. 4. This demonstrates that a combination therapy approach that integrates shRNA into CAR-expressing retroviral vectors, while simultaneously gene expression and modulation, can be achieved, with simultaneous expression of LSD1shRNA and anti-CD19 CAR.

Example in vitro study of four LSD1shRNA enhancement of anti-tumor function of anti-CD19CAR-T cells

LSD1shRNA enhanced anti-CD19CAR-T cell in vitro killing tumor cell function detection

1.1.LSD1 shRNA anti-CD19CAR-T cell in vitro killing Raji cell efficiency detection

Co-culturing LSD1 shRNA-1 anti-CD19CAR-T cells and tumor cells Raji positive to CD19 molecule expression for 12 hours according to the number ratio of 1:16,1:8,1:4,1:2 and 1:1, and detecting the efficiency of killing Raji cells by the LSD1shRNA anti-CD19CAR-T cells by flow cytometry. The results show that: the killing efficiency of LSD1 shRNA-1 anti-CD19CAR-T cells is obviously enhanced, and panT refers to untransformed T cells, as shown in FIG. 5.

1.2LSD1 shRNA anti-CD19CAR-T cells in vitro killing Raji cell pressure test detection CAR-T cells and Raji cells were co-cultured at a number ratio of 1:1 for 48 h. Flow cytometry detects the expression of CD19 molecules of the co-cultured cells, and as a result, the LSD1shRNA anti-CD19CAR-T cell group and the RNAU6 anti-CD19CAR-T cell group are found to have no cells positive for the expression of the CD19 molecules. Repeating the co-culture for 3 cycles, and after each co-culture cycle is finished, carrying out apoptosis detection experiments of killing Raji cells by the CAR-T cells, and detecting the killing efficiency of killing the Raji cells by the CAR-T cells in vitro by using a flow cytometry method for 3 times.

After 48h of co-culture 1, apoptosis detection experiments of the CAR-T cell killing Raji cells find that: the killing efficiency of the LSD1shRNA anti-CD19CAR-T cells and the RNAU6 anti-CD19CAR-T cells for killing Raji cells is enhanced compared with that of the LSD1shRNA anti-CD19CAR-T cells without antigen stimulation, and the killing efficiency of the LSD1shRNA anti-CD19CAR-T cells is obviously enhanced compared with that of the RNAU6 anti-CD19CAR-T cells, as shown in fig. 6A.

After 48h of co-culture 2, apoptosis detection experiments of the CAR-T cell killing Raji cells find that: the killing efficiency of the LSD1shRNA anti-CD19CAR-T cells and the RNAU6 anti-CD19CAR-T cells for killing Raji cells is not obviously different from the killing efficiency of the LSD1shRNA anti-CD19CAR-T cells after the 1 st co-culture period, and the killing efficiency of the LSD1shRNA anti-CD19CAR-T cells is obviously enhanced compared with the RNAU6 anti-CD19CAR-T cells, as shown in figure 6B.

After 48h of co-culture at 3 rd time, apoptosis detection experiments of CAR-T cell killing Raji cells found that: the killing efficiency of the LSD1shRNA anti-CD19CAR-T cells and the RNAU6 anti-CD19CAR-T cells for killing Raji cells is reduced compared with that after the 2 nd co-culture period, which indicates that the CAR-T cells can be gradually depleted under the stimulation of long-term tumor antigens, and the efficiency of the LSD1shRNA anti-CD19CAR-T cells is still obviously enhanced compared with that of the RNAU6 anti-CD19CAR-T cells, which indicates that the LSD1shRNA can be beneficial to the long-term anti-tumor function of the anti-CD19CAR-T cells, as shown in fig. 6C and the following table.

1.3LSD1 shRNA anti-CD19CAR-T cell in vitro killing Raji-Luc cell efficiency detection

LSD1shRNA anti-CD19CAR-T cells are co-cultured with Raji-Luc cells stably expressing luciferase, and the luciferase detects the killing efficiency of the CAR-T cells. The results show that: compared with RNAU6 anti-CD19CAR-T cells, the efficiency of killing Raji-Luc cells by the two groups of LSD1shRNA anti-CD19CAR-T cells is obviously enhanced, and the efficiency is shown in FIG. 7 and the following table.

1.4LSD1 shRNA anti-CD19CAR-T cell killing SW620 cell efficiency assay.

Inoculation of RTCA-matched E-plate 96 assay plates with 1X 10 ⁴ After 48h of detection recording, 2500 PanT or RNAU6 anti-CD19CAR-T or LSD1 shRNA-1 anti-CD19CAR-T cells (effective target ratio 1:4) were added and recording continued for 3 days. After the detection is finished, the data analysis result shows that: the SW620 cell index of the LSD1 shRNA-1 anti-CD19CAR-T cell group is obviously lower than that of the RNAU6 anti-CD19CAR-T cell control group, and statistical differences exist, see FIG. 8 and the table below, which show that the killing efficiency of the SW620 cell killing of the LSD1 shRNA-1 anti-CD19CAR-T cell is obviously enhanced.

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1.5LSD1 shRNA anti-CD19CAR-T cell cytokine release level detection

ELISA detects cytokine release level in cell supernatant after co-culturing LSD1shRNA anti-CD19CAR-T cells and target cells Raji for 12 h. The result shows that: the levels of IFN-gamma, TNF-alpha, L-2 release were all significantly increased in the cell supernatants, see FIG. 9 and the following tables.

Detection of LSD1shRNA enhanced anti-CD19CAR-T cell in vitro proliferation capacity

2.1CFSE proliferation assay

After staining RNAU6 anti-CD19CAR-T cells, LSD1 shRNA-1 anti-CD19CAR-T cells and LSD1 shRNA-2 anti-CD19CAR-T cells with CFSE, taking the stained cells for flow cytometry analysis. The results show that: CFSE staining was relatively uniform among groups, and FITC signal average fluorescence intensity was not significantly different.

The CAR-T cells with uniform CFSE staining are respectively cultured for 24 hours under the condition of target cell antigen stimulation (the number ratio of effector cells to target cells is 1:2), and the proliferation condition of the CAR-T cells is detected by flow cytometry. The results show that: compared with RNAU6 anti-CD19CAR-T cells, the average fluorescence intensity of FITC signals of the two groups of LSD1shRNA anti-CD19CAR-T cells is obviously reduced, namely the CFSE signal intensity is obviously reduced, and the proliferation speed of the LSD1shRNA anti-CD19CAR-T cells is obviously faster.

2.2 cell proliferation recordings.

Cells were cultured continuously for 20 days, counted every 48 hours, and passaged to a concentration of 1X 10 ⁶ And each mL. And calculating the cell growth fold according to the cell count, and preparing an LSD1shRNA anti-CD19CAR-T cell proliferation curve and a survival rate curve. It can be seen that as the cell culture time was prolonged, the proliferation capacity and persistence of LSD1shRNA anti-CD19CAR-T cells were enhanced after day 15 compared to RNAU6 anti-CD19CAR-T cells, see FIG. 10 and the following table.

2.3 CD4 in CAR-T cells ⁺ T cells and CD8 ⁺ T cell detection

When only effector cells are present, CD4 in RNAU6 anti-CD19CAR-T cells and LSD1shRNA anti-CD19CAR-T cells ⁺ T cells and CD8 ⁺ T cell ratio was not significantly different, LSD1shRNA was not specific for CD4 ⁺ T cells and CD8 ⁺ The ratio change of the T cells has obvious influence; after the effector cells and target cells Raji are co-cultured for 12 hours, CD8 in the RNAU6 anti-CD19CAR-T cells and LSD1shRNA anti-CD19CAR-T cells ⁺ T cells were all significantly increased, with no significant differences between groups, see FIG. 11.

2.4TCM cell detection

TCM cells expressed CD45RO and CD62L molecules positively, and flow cytometry detected TCM cell content changes in LSD1shRNA anti-CD19CAR-T cells, i.e., the proportion of CD45RO and CD62L biscationic cell populations in the CD3 positive cell population was changed, as a result of which it was found that: the TCM cell content in LSD1shRNA anti-CD19CAR-T cells is not obviously different from the TCM cell content in the RNAU6 anti-CD19CAR-T cells of the control group.

Example five LSD1shRNA in vivo study of enhancing anti-tumor function of anti-CD19CAR-T cells

1. Construction of tumor animal model

Tumor signals were visualized by in vivo imaging of animals 4 days after intravenous injection of Raji-Luc cells into the tail of NPG mice, and the average photon count was (1.53.+ -. 0.34). Times.10 ⁴ Indicating that the tumor animal model is successfully constructed.

2. Tumor monitoring in vivo imaging of small animals

The results of the in vivo imaging detection of the small animals show that: the tumor signals of mice in the model group and the PanT-treated group injected with the non-transduced CAR were gradually increased and died successively, whereas the tumor signals of mice in the RNAU6 anti-CD19CAR-T cell treated group and the LSD1shRNA anti-CD19CAR-T cell treated group disappeared, and no tumor signal was detected by day 52 after the end of the experiment, and the tumor area size and the overall signal intensity change are shown in fig. 12.

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3.3 recording mice survival curves

The mice survived by tail vein injection of Raji-Luc cells on day 0 and continuous monitoring until day 52, and the survival time of the mice in the CAR-T cell treatment group was obviously prolonged, as shown in FIG. 13 and the following table.

3.4 mice weight detection

Mice were monitored for weight changes, and it was seen that model and PanT mice lost weight sharply around day 20 after tumor cell injection, and subsequently died. Whereas the CAR-T cell treated mice had a smoother body weight change and had a tendency to rise slowly, see figure 14 and table below.

Weight change of mice (g)

3.5 in vivo T cell proliferation level detection CD3 was consistently detected in peripheral blood of mice of the CAR-T cell treatment group ⁺ T until the experiment ended. On day 52 after injection of tumor cells Raji-Luc into NPG mice, T cell content was detected using BV 785-labeled anti-human CD3 antibody flow cytometry, anti-CD19CAR-T cell treated group mice peripheral blood CD3 ⁺ The ratio of the T cells to the nucleated cells in the peripheral blood is (1.29+/-0.99)%; RNAU6 anti-CD19CAR-T cell treatment group was (1.82+ -0.94)%; the LSD1 shRNA-1 anti-CD19CAR-T cell treatment group is (5.32+/-1.17)%, and the LSD1 shRNA-2 anti-CD19CAR-T cell treatment group is (9.52+/-5.23)%. The T cell number was calculated from the standard curve, namely: each 100 mu L of mouse peripheral blood contains 5187.04 +/-8329.14 anti-CD19CAR-T cells; 7181.21 + -7951.34 RNAU6 anti-CD19CAR-T cells; 38597.9 + -9925.14 LSD1 shRNA-1 anti-CD19CAR-T cells; LSD1 shRNA-2 anti-CD19CAR-T cells 72228.47 + -44207.59. The content of T cells in peripheral blood of mice in the LSD1shRNA anti-CD19CAR-T cell treatment group is obviously higher than that in the RNAU6 anti-CD19CAR-T cell treatment group.

3.6 detection of IFN-gamma Release level in mouse serum

ELISA detects IFN-gamma release levels in NPG mice serum 7 days after the second injection of CAR-T cells. The results show that the release level of IFN-gamma in the serum of mice in the LSD1shRNA anti-CD19CAR-T cell treatment group is obviously increased, and the results are shown in FIG. 15 and the following table.

It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.

Sequence listing

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Claims

1. A retroviral vector expressing a CAR and a shRNA, wherein the shRNA targeting LSD1 in the viral vector is co-expressed with the CAR; the retrovirus vector comprises a U6 promoter, an LSD1shRNA, an EF1 alpha promoter, an upstream signal peptide and a myc tag for detection which are sequentially connected in series; a CD19CAR antigen binding region; CD8 hinge-transmembrane domain; a CD28 or 4-1BB co-activation domain and a cd3ζ intracellular signaling domain; the clone ID of shRNA targeting LSD1 is TRCN0000046068, the clone name is NM_015013.1-1812s1c1, and the sequence is SEQ ID NO. 2: GCCTAGACATTAAACTGAATA; or the clone ID of shRNA targeting LSD1 is TRCN0000046069, the clone name is NM_015013.1-2168s1c1, and the sequence is SEQ ID NO. 3: GCTCCAATACTGTTGGCACTA.

2. A targeted chimeric antigen receptor T cell comprising a targeted chimeric antigen receptor expressed by the retroviral vector of claim 1.

3. A medicament for the treatment of tumors, characterized in that it contains the chimeric antigen receptor T cells according to claim 2.

4. A medicament according to claim 3, characterized in that the tumour is multiple myeloma.